Histone Messenger Research Articles

Length suppression in histone messenger RNA 3'-end maturation: processing defects of insertion mutant premessenger RNAs can be compensated by insertions into the U7 small nuclear RNA.

Efficient 3'-end processing of cell cycle-regulated mammalian histone premessenger RNAs (pre-mRNAs) requires an upstream stem-loop and a histone downstream element (HDE) that base pairs with the U7 small ribonucleoprotein. Insertions between these elements have two effects: the site of cleavage moves in concert with the HDE and processing efficiency declines. We used Xenopus oocytes to ask whether compensatory length insertions in the human U7 RNA could restore the fidelity and efficiency of processing of mouse histone insertion pre-mRNAs. An insertion of 5 nt into U7 RNA that extends its complementary to the HDE compensated for both defects in processing of a 5-nt insertion substrate; a noncomplementary insertion into U7 did not. Yet, the noncomplementary insertion mutant U7 was shown to be active on insertion substrates further mutated to allow base pairing. Our results suggest that the histone pre-mRNA becomes rigidified upstream of its HDE, allowing the bound U7 small ribonucleoprotein to measure from the HDE to the cleavage site. Such a mechanism may be common to other RNA measuring systems. To our knowledge, this is the first demonstration of length suppression in an RNA processing system.

The beneficial effects of incubating bovine embryos in groups are due to platelet-derived growth factor

This report presents an analysis of histone gene expression in the cleaving embryo of the sea urchin, Strongylocentrotus purpuratus, with emphasis on whether the regulatory site(s) in the pathway of gene expression change as development proceeds. The analysis focuses on the equation, dP∗dt = M·f·n·At, where dP∗dt = the absolute rate of histone synthesis; M = the mole quantity of histone messenger RNA; f = the fraction of histone mRNA in polysomes; n = the polysome size; and At = the rate of elongation of nascent histone polypeptide chains. The embryo solves this rate equation differently at different times. Measurements were made (at 15°C) of absolute rates of histone synthesis (dP∗dt). The rate of histone synthesis increases at least 48-fold during the first 6 hr after fertilization from less than 0.5 to 24 pg embryo−1 hr−1; in the period from 6 to 12 hr, this rate rises to 182 pg embryo−1 hr−1, an additional 7.7-fold rise, resulting in an overall increase of 370-fold between the 1-cell and 200-cell stage. The fraction of newly synthesized (zygotic) histone messenger RNA that partitions into polysomes (fzygotic) has also been measured during the first 12 hr of development. This fraction increases from 0.2 in the 2-hr embryo to 0.8 in the 6-hr embryo (16-cell stage), increasing slowly thereafter to near unity by 12 hr. The size of histone-synthesizing polysomes (n) does not change substantially over the 12-hr interval, remaining constant at a weighted mean of 5 ribosomes per polysome (range 3 to 7). Utilizing the data on fzygotic and dP∗dt, the rate of elongation of nascent histone polypeptide chains (At) during the first 6 hr of development was estimated; At remains constant at 1.11 codons per second. This calculated value is in fair agreement with a direct measurement of histone peptide elongation rate in the 12-hr embryo. It is proposed that histone gene expression in cleaving sea urchin embryos be divided into two phases, distinguished on the basis of their pivotal translational parameters: Phase I (0–6 hr), during which f is rate determining, and Phase II (6 hr on), during which M is the rate-determining parameter.

Efflux of RNA from Resealed Nuclear Envelope Ghosts

mRNA translocation across the nuclear envelope and the appropriate signal-receptor interactions have been studied using resealed rat liver nuclear envelope ghosts (RNEG). We compared export kinetics of nonadenylated (tRNAs, histone-2 poly(A) − mRNA), and adenylated RNAs (poly(A) + tRNAs, synthetic histone-2 poly(A)+mRNA, albumin mRNA, β-globin poly(A) +mRNA and a total poly(A) + mRNA extract from rat liver cells). ATP-dependent export of mRNAs and of total poly(A) (+) RNA was prevented by inhibitors of a nuclear envelope NTPase. All adenylated RNA species competed with each other for export, but nonadenylated RNAs did not. This indicates the existence of different translocation mechanisms for different RNA species with their appropriate nuclear envelope associated RNA receptors involved in export. The attachment of a poly(A) 250 sequence at the 3′-end of tRNA or histone messenger masks the intrinsic RNA export signal of nonadenylated RNAs and results in efflux comparable to that of β-globin poly(A) + mRNA. The attachment on oligo(A) 5 does not have any comparable effect of nonadenylated RNA translocation. Export of all polyadenylated RNAs from RNEGs is blocked by a monoclonal antibody, which is directed against an intranuclear envelope poly(A) binding protein. The results suggest that the pore complexes do not select RNAs for export to the cytoplasm and are therefore not responsible for nuclear restriction of mRNA precursors.

Analysis of hepatocellular proliferation: study of archival liver tissue is facilitated by an endogenous marker of DNA replication.

Assessment of liver regeneration with endogenous genes that are expressed during DNA replication is physiological, specific and direct. To determine whether H3 histone messenger RNA expression (which is tightly coupled with DNA synthesis) could be used for this purpose, we initially examined liver regeneration in a mouse model. After partial hepatectomy, RNA transblot studies showed induction of H3 histone messenger RNA expression in regenerating mouse livers. In situ molecular hybridization demonstrated that the overall pattern of H3 histone messenger RNA expression correlated with [3H]thymidine labeling of hepatocytes. After partial hepatectomy, H3 histone messenger RNA expression in hepatocytes peaked at 48 hr (greater than 60 times greater than at 24 hr; p less than 0.001) and then rapidly declined. Although hepatocyte labeling with [3H]thymidine showed similar kinetics of liver regeneration, use of this parameter resulted in overestimation of the proliferative compartment when it was compared with H3 histone messenger RNA expression. Next we determined whether H3 histone messenger RNA expression could be used to study hepatocellular proliferation in archival human material. H3 histone messenger RNA-expressing hepatocytes were identified on in situ hybridization in patients with acute or chronic active hepatitis and active cirrhosis, but not inactive cirrhosis. These studies demonstrate that H3 histone messenger RNA is expressed in a phasic manner during liver regeneration. Use of H3 histone messenger RNA expression to evaluate hepatocellular proliferation should facilitate clinical studies and greatly advance our understanding of the pathophysiology of liver regeneration.

Cell cycle-dependent regulation of histone precursor mRNA processing by modulation of U7 snRNA accessibility

Histone gene expression is regulated both during and after transcription, with the turnover of histone messenger RNA and the regulation of its 3' processing being the principal factors that determine the size of the histone mRNA pool during the cell cycle. The mature ends of the replication-dependent histone mRNAs are generated during 3' processing by endonucleolytic cleavage of a large primary transcript. This reaction depends on a highly conserved stem-loop structure also included in the mature RNA, and a purine-rich sequence lying downstream within the spacer transcript. Three factors involved in this reaction which act in trans have been identified: the U7 small nuclear ribonucleoprotein particle (snRNP), the heat-labile factor, and the hairpin-binding factor. Here we show how U7 snRNP participates in the regulation of 3' processing: the 5' sequences of the U7 snRNA that hybridize with the downstream spacer motif during 3' processing are occluded in the G0 stage of the cell cycle but are exposed and free to interact with histone pre-mRNA during S phase, when histone mRNA synthesis is at its peak.

Sequence, organization and expression of the core histone genes ofAspergillus nidulans

The core histone gene family of Aspergillus nidulans was characterized. The H2A, H2B and H3 genes are unique in the A. nidulans genome. In contrast there are two H4 genes, H4.1 and H4.2. As previously reported for the H2A gene (May and Morris 1987) introns also interrupt the other core histone genes. The H2B gene, like the H2A gene, is interrupted by three introns, the H3 and H4.1 gene are each interrupted by two introns and the H4.2 gene contains one intron. The position of the single intron in H4.2 is the same as that the first intron of the H4.1 gene. The H2A and H2B genes are arranged as a gene pair separated by approximately 600 bp and are divergently transcribed. The H3 and H4.1 genes are similarly arranged and are separated by approximately 800 bp. The H4.2 gene is not closely linked to either the H2A-H2B or H3-H4.1 gene pairs. Using pulse field gel electrophoresis an electrophoretic karyotype was established for A. nidulans. This karyotype was used to assign the H3-H4.1 gene pair and the H4.2 gene to linkage group VIII and the H2A-H2B gene pair to either linkage group III or VI. The abundance of each of the histone messenger RNAs was determined to be cell cycle regulated but the abundance of the H4.2 mRNA appears to be regulated differently from the others.

Estrogen effects on histone messenger ribonucleic acid levels in the rat uterus.

RNA was isolated from uteri of immature rats before and after estrogen treatment. The concentration of histone mRNA was analyzed by Northern hybridization and compared with messenger RNA concentration of alpha-actin, beta-actin, and beta-tubulin. Steady state levels of common histone mRNAs did not change up to 9 h after hormone administration. After that time the histone mRNA levels increased significantly and reached a maximum at 18 h, several hours later than the time of maximal histone protein biosynthesis induced by estrogen. The concentration of control mRNAs (alpha- and beta-actin and beta-tubulins) increased shortly after estradiol injection and reached a peak at 9 h. These results show that the pattern of histone gene expression induced by estrogen has some features similar to those observed during embryogenesis.

Regulation of human histone gene expression: transcriptional and posttranscriptional control in the coupling of histone messenger RNA stability with DNA replication.

The extent to which transcriptional and posttranscriptional regulation contributes to the coupling of histone gene expression and DNA replication was examined during the cell cycle in synchronized HeLa S3 cells. Rates of transcription were determined in vitro in isolated nuclei. A 3-5-fold increase in cell cycle dependent histone gene transcription was observed in early S phase, prior to the peak of DNA synthesis. This result is consistent with a previous determination of histone mRNA synthesis in intact cells [Plumb, M., Stein, J., & Stein, G. (1983) Nucleic Acids Res. 11, 2391]. The transcription of these genes did not change appreciably after inhibition of DNA replication by hydroxyurea treatment, although Northern blot analysis indicated that cellular levels of histone mRNA decreased rapidly in the presence of the drug. Total cellular levels of histone mRNA closely parallel the rate of DNA synthesis as a function of cell cycle progression, reaching a maximal 20-fold increase as compared with non S phase levels. This DNA synthesis dependent accumulation of histone mRNA occurs predominantly in the cytoplasm and appears to be mediated primarily by control of histone mRNA stability. Changes in nuclear histone mRNA levels were less pronounced. These combined observations suggest that both transcriptional regulation and posttranscriptional regulation contribute toward control of the cell cycle dependent accumulation of histone mRNA during S phase, while the stability of histone mRNA throughout S phase and the selective turnover of histone mRNAs, either at the natural termination of S phase or following inhibition of DNA synthesis, are posttranscriptionally regulated.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular characterization of the histone gene family of Caenorhabditis elegans

The core histone genes (H2A, H2B, H3 and H4) of Caenorhabditis elegans are arranged in approximately 11 dispersed clusters and are not tandemly arrayed in the genome. Three well-characterized genomic clones, which contain histone genes, have one copy of each core histone gene per cluster. One of the clones (λCeh-1) carries one histone cluster surrounded by several thousand base-pairs of non-histone DNA, and another clone (λCeh-3) contains a histone cluster duplication surrounded by non-histone DNA. A third clone (λCeh-2) carries a cluster of core histone genes flanked on one side (12,000 base-pairs away) by a single H2B gene and on the other by non-histone DNA. A fourth cluster (clone BE9) has one copy each of H3 and H4 and two copies each of H2A and H2B. This cluster is also flanked by non-histone DNA. Analysis of cosmid clones which overlap three of the clusters shows that no other histone clusters are closer than 8000 to 60,000 base-pairs, although unidentified non-histone transcription units are present on the flanking regions. Gene order within the histone clusters varies, and histone mRNAs are transcribed from both DNA strands. No H1 sequences are found on these core histone clones. Restriction fragment length polymorphisms between two related nematode strains (Bristol and Bergerac) were used as phenotypic markers in genetic crosses to map one histone cluster to linkage group V and another to linkage group IV. Hybridization of gene-specific probes from sea urchin to C. elegans RNA identifies C. elegans core histone messenger RNAs of sizes similar to sea urchin early stage histone mRNAs (H2A. H2B, H3 and H4). The organization of histone genes in C. elegans resembles the clustering found in most vertebrate organisms and does not resemble the tandem patterns of the early stage histone gene family of sea urchins or the major histone locus of Drosophila.

Coordinate turnover of nuclear and cytoplasmic histone messenger RNA following inhibition of DNA replication in HeLa S3 cells.

We have examined the metabolism of human H4 histone mRNA in the nucleus and cytoplasm of HeLa S3 cells following inhibition of DNA synthesis to address the extent to which histone mRNA stability in these cellular compartments is coupled to DNA replication. The nuclear and cytoplasmic levels of histone mRNAs encoded by the pF0108A human H4 histone gene were determined by S1 nuclease analysis using a 32P-labeled probe that could distinguish pF0108A transcripts from those of other members of the H4 histone multigene family. Hydroxyurea treatment resulted within 15 min in a 75% reduction in the level of histone H4 mRNA in the nucleus, which corresponds to the 85% decrease observed for H4 histone mRNA in the cytoplasm. The kinetics of nuclear and cytoplasmic H4 mRNA turnover following hydroxyurea treatment were also similar. Northern blot analysis using a 32P-labeled mitochondrial cytochrome b probe indicated that the association of cytoplasmic RNA with the nuclear fraction was less than 0.5%. Treatment of cells with a protein synthesis inhibitor resulted in a 1.3-fold increase in nuclear H4 histone mRNA levels and a 1.5-fold increase of H4 mRNA in the cytoplasm after 45 min. Together, these results indicate that nuclear and cytoplasmic H4 histone mRNAs respond similarly to metabolic perturbations that influence message stability and that mechanisms operative in the turnover of histone mRNAs in the nucleus and cytoplasm may be similar.

Histone messenger RNAs of the mouse testis

A 6-12S RNA fraction has been isolated following sucrose gradient fractionation of mouse testis RNA, and further resolved into poly A+ and poly A- RNA fractions by oligo-(dt)-cellulose chromatography. Polyacrylamide gel electrophoresis of products formed in a reticulocyte lysate-dependent cell-free translation system has enabled identification of histone variants, Hlt, H2S, H2A-X, an H4-like protein and a low M r protein (presumably TP and/or protamine). Cell-free synthesis of a number of these histone variants appears to be directed by poly A+ mRNAs.

H4 histone messenger RNA decay in cell-free extracts initiates at or near the 3′ terminus and proceeds 3′ to 5′

The relative decay of four human messenger RNAs, gamma globin, delta globin, c-myc and H4 histone, were compared in a cell-free system. Under appropriate conditions, they are degraded in vitro in approximately the same relative order as in vivo: histone faster than c-myc and delta globin faster than gamma globin. Degradation of polysome-associated H4 histone mRNA and of deproteinized histone mRNA begins at or near the 3' terminus. At least a portion of the mRNA then continues to be degraded in a 3' to 5' direction. Discrete 3'-terminal degradation hold-up points are observed, suggesting that 3' to 5' degradation occurs non-uniformly. Cycloheximide and puromycin inhibit protein synthesis but do not affect the rate or directionality of histone mRNA decay in vitro. We conclude that the rate-limiting step in H4 histone mRNA decay occurs at or near the 3' terminus and that at least a portion of the mRNA molecule is subsequently degraded 3' to 5', probably via a processive exonuclease.

Simultaneous expression of early and late histone messenger RNAs in individual cells during development of the sea urchin embryo

The transition from early (E) to late (L) histone gene expression in developing sea urchin ( Strongylocentrotus purpuratus) embryos was examined for H2B, H3, and H4 mRNAs by in situ hybridization of class-specific probes. Hybridization patterns indicate that the shift from E to L mRNAs occurs gradually and simultaneously in all blastomeres. Thus, during the transition the ratio of L to E mRNAs is similar in most cells. This suggests that no sudden changes in histone composition occur in individual cells which might be related to alterations in gene expression associated with differentiation of cell lineages. Around the midpoint of the transition, clusters of cells progressively appear which contain little, if any, E or L histone mRNA. This modulation of expression is coordinated for the three late genes examined because most individual cells contain either high or low levels of all three mRNAs. At blastula stage these clusters of unlabeled cells appear to be randomly distributed throughout the embryo. Subsequently the unlabeled regions expand and are found predominantly in aboral ectoderm as these cells cease to divide. Thus, the L E histone mRNA ratio is not differentially regulated in diverse cell lineages, and the major differences in total histone mRNA content among individual cells may be related to cell cycle and/or the cessation of division.

Subcellular localization of histone messenger RNAs on cytoskeleton-associated free polysomes in HeLa S3 cells.

We have examined the subcellular distribution of histone mRNA-containing polysomes in HeLa S3 cells to assess the possible relationship between localization of histone mRNAs and the regulation of cellular histone mRNA levels. The distribution of histone mRNAs on free and membrane bound polysomes was examined as well as the association of histone mRNA-containing polysomes with the cytoskeleton. The subcellular localization of histone mRNAs was compared with that of HLA-B7 mRNAs which encode a cell surface antigen. Histone mRNAs were localized predominantly on the free polysomes, whereas the HLA-B7 mRNA was found almost exclusively on membrane bound polysomes. However, both species of mRNA were found associated with the cytoskeleton. Interruption of DNA synthesis by hydroxyurea treatment resulted in a rapid and selective destabilization of histone mRNAs in each subcellular fraction; in contrast, the stability of HLA-B7 mRNA appeared unaffected. The results presented confirm that histone mRNAs are predominantly located on non-membrane bound polysomes and suggest that these polysomes are associated with the cytoskeletal framework.

Histone messenger RNA synthesis and accumulation during early development in the echiuroid worm, Urechis caupo

RNA isolated from Urechis caupo mature oocytes and embryos was analyzed for the presence of histone messenger RNAs (mRNAs) by in vitro translation and by filter blot hybridization to determine the contribution of maternal and newly transcribed histone mRNAs to the pattern of histone synthesis during early development. Histone mRNAs were not detected in mature oocyte RNA which suggests that relatively few if any maternal histone mRNAs are sequestered in the mature oocytes. Histone mRNAs were detected in cleavage-stage RNA and increased in amount from midcleavage through late gastrula stages. The in vitro translation analysis also demonstrated that the amount of H1 histone mRNA in late cleavage- and early blastula-stage embryos exceeds that of the individual core histone mRNAs. The disproportionate accumulation of individual histone mRNAs correlates with the noncoordinate synthesis of H1 and core histones which occurs during early embryogenesis.

Histone H4 gene is transcribed in S phase but also late in G2 phase in Physarum polycephalum

The myxomycete Physarum polycephalum contains two types of H4 histone genes. Southern blotting of restriction endonuclease fragments of P. polycephalum DNA and hybridization to a cloned probe labelled by nick-translation indicate that there are only one or two copies of each H4 gene per haploid genome. A cloned homologous genomic probe was used to study the cellular abundance of H4 mRNA during the cell cycle. We report that the H4 mRNA is not only transcribed in S phase as previously described for other organisms but that transcription of the H4 gene also occurs at the end of G(2) phase. Since no translation of the histone messenger was observed in G(2) phase this suggests that the histone mRNA synthesized in G(2) constitutes a pool of molecules in anticipation of the next S phase.

Temporal expression of late histone messenger RNA in the sea urchin Lytechinus pictus.

Sea urchin histones are encoded by several multigene families. The temporal expression of one of these families, the late histones, has been studied during the early development of Lytechinus pictus. Using a nuclease S1 assay, we detected about 10,000 transcripts encoding both late H3 and H4 proteins in the unfertilized egg. This suggests that the late genes were active at some point during oogenesis. The number of late gene transcripts begins to increase 6.5 hr after fertilization (64-cell stage), indicating that these genes probably become reactivated 4.5-6.5 hr after fertilization. The maximum rate of accumulation of transcripts (4600 molecules per min per embryo) occurs 9-14 hr after fertilization (from blastula stage to hatching). The number of transcripts peaks 21 hr after fertilization (onset of gastrulation) when the embryo has accumulated 1.8 X 10(6) copies of each late mRNA (a 164-fold increase). A 5.5-fold increase in the relative rate of transcription, between 7 and 15 hr after fertilization, is partly responsible for the accumulation of these gene products. The relative synthesis of early histone message, which is encoded by a different family, decreases 18-fold during this time. Synthesis of the late transcripts continues at the higher rate after accumulation has ceased (24 hr after fertilization). The number of late transcripts begins to decrease 48 hr after fertilization, reaching about 10,000 copies at 72 hr.

Processing and nucleo-cytoplasmic transport of histone gene transcripts.

Precursors of Xenopus and sea urchin histone mRNAs were synthesized in vitro with the SP6 transcription system, and their maturation and nucleo-cytoplasmic transport was studied by frog oocyte injection. 3' processing is most rapid for homologous histone messenger sequences and does not require either genuine 5' or specific 3' ends of the precursor, but capping of the 5' terminus strongly influences the efficiency of 3' processing. No generation of 5' histone mRNA ends can be detected when precursors containing 5' spacer sequence extensions are injected into the oocyte nucleus. This finding may have some implications for the question whether histone gene transcription could be polycistronic. Using a novel oocyte technique, we have separated nuclei from cytoplasm and have studied the time course of exit of the processed RNA from the oocyte nucleus into the cytoplasm. The results suggest that RNA maturation and nucleo-cytoplasmic transport are not temporally coupled processes.

Yeast H3 and H4 histone messenger RNAs are transcribed from two non-allelic gene sets

The genes coding for the H3 and H4 histones of Saccharomyces cerevisiae have been isolated by recombinant DNA cloning. The genes were detected in a bacteriophage lambda library of the yeast genome by hybridization with plasmids containing the cloned Psammechinus miliaris sea urchin histone genes (pCH7) and the cloned Drosophila histone genes (cDM500). Two non-allelic sets of the H3 and H4 genes have been isolated. Each set consists of one H3 gene and one H4 gene arranged as a divergently transcribed pair separated by an intergene spacer DNA. The histone genes were located on the cloned yeast fragments by S1 nuclease mapping, as was a gene (SMT1) of unknown function that does not code for a histone but is closely linked to one of the histone sets. Sequence homology between the two non-allelic sets is confined to the coding regions of the respective genes while the flanking DNA and intergene spacer DNA are extensively divergent. Cellular RNA homologous to the histone genes, including transcribed non-coding sequences unique to each of the four genes, was detected by S1 mapping, thus demonstrating that all four genes are transcribed in vegetative cells.

The lack of cell contact causes genomic activation in early sea urchin embryos

If Paracentrotus Lividus embryos at the 4–8 cell stage are dissociated into cells and prevented from reaggregating, an increase of at least 270% is observed in the RNA synthesis. Preliminary analyses indicate that the newly synthesized RNA is ribosomal RNA and histone messenger.